Plasma long-chain fatty acids are the primary fuel source for energy production in the normal heart. In the ischemic and reperfused myocardium, there is decreased utilization of fatty acids, and perfusion with fatty acids results in in situ membrane damage and cardiac dysfunction. Fatty acid-induced injury to the ischemic myocardium can be reduced by inhibition of carnitine palmitoyltransferase I (CPTI), a rate-limiting enzyme in beta-oxidation. For effective pharmacotherapy of defects in cardiac fatty acid oxidation, it is imperative that we understand the biochemical and molecular mechanisms regulating M-CPTI, a key enzyme in myocardial bioenergetics. Our working hypothesis, supported by recent mutagenesis studies, is that amino acids essential for malonyl- CoA inhibition and binding and for substrate binding and catalysis in M-CPTI reside in the N- and C-terminal regions, respectively. Furthermore, because of the essential role of M- CPTI in heart function, loss of the enzyme may result in death. Our specific aims are: (1) To identify specific amino acid residues important for malonyl-CoA inhibition and binding by deletion and substitution mutation analysis of chimeric constructs of M-CPTI and L-CPTI, and by cysteine scanning mutagenesis. (2) To map the substrate binding and catalytic site pocket of M-CPTI by site-directed mutagenesis, ligand binding, and intrinsic tryptophan fluorescence quenching studies. (3) To purify milligram quantities of P. pastoris- and E. coli-expressed M-CPTI and engineered fragments for structural characterization. (4) To generate a heart-specific conditional knockout mouse model for M-CPTI using the Cre-loxP system, and to determine its effect on embryonic lethality. The M-CPTI null mice will allow us to construct models that mimic human CPT deficiency diseases and to look for potential gene therapy through retro-transfection of the normal CPT gene.